Project Summary There have been many recent successes in the treatment of autoimmune and inflammatory diseases including the development of antibodies and biologic proteins that block the pathogenic functions of inflammatory cytokines such as TNF and IL-17. However, it remains a challenge to achieve sustained responses with these anti-inflammatory therapeutics. We and others have attempted to achieve this goal through the development of inhibitory receptor agonists that limit the activation of pathogenic lymphocytes, and that potentially alter the long-term differentiation of autoreactive cells. The BTLA inhibitory receptor has proven to be an important regulatory receptor in lymphocytes that we have shown can be triggered using agonist antibodies and biologic proteins. We have previously developed a human BTLA-specific agonist protein through site-directed mutagenesis of the BTLA ligand (HVEM) that does not bind other cellular factors, and that shows greater affinity and inhibitory activity in lymphocytes in vitro. In vivo analysis of this protein is limited due to species restriction of the human HVEM protein. Here, we have proposed to test this protein using a xenogenic humanized GVHD model in which human PBMC from normal donors are transferred in to Nod-scid IL2R?-/- hosts. This model has proven to be a robust system to evaluate human immune responses in vivo. We will confirm activity of our engineered protein in human cells, and determine the efficacy of this protein in the humanized GVHD model. Additionally we will further evaluate the molecular and cellular determinants that are required for this protein to achieve optimal efficacy. Completion of the aims in this proposal represent an important step in the validation of this protein as a potential human anti-inflammatory therapeutic, and will allow us to pursue further clinical development. Additionally this proposal will provide further insight into how the BTLA inhibitory receptor functions to regulate human immune responses in vivo.